Gliomas
The major cell types of the brain are neurons, oligodendrocytes, which are the cells forming the myelin sheaths around neuronal axons in the Central Nervous System (CNS), and astrocytes, the cells supporting both neuron and oligodendrocyte functions. Oligodendrocytes and astrocytes, together with ependymal cells that line the ventricular system, constitute the glia, the specialised connective tissue of the CNS.
Gliomas are tumours that show predominant glial differentiation and are the most frequent subtype of primary brain tumours, with an incidence of 5-10 per 100.000 in the general population per year. The vast majority of gliomas are of sporadic origin, but some genetic diseases have been associated with an increased predisposition to their development like Neurofibromatosis type 1(a familial syndrome characterised by mutations in the NF1 tumour suppressor gene), Li-Fraumeni (inherited germ-line mutations in the TP53 gene) and Turcot syndromes (a familial syndrome characterised by polyposis cancers linked to inherited mutations in the APC gene). The severity of gliomas appears absolutely obvious given the fundamental role of the brain and the damages that can be caused by neurological destruction. Furthermore, even the so-called benign forms are in reality most often lethal, mainly because of the high propensity of these tumours to spread into normal brain structures and to progress towards malignancy. Therefore, gliomas are considered to be among the most devastating of human malignancies, and despite progress in therapy, including surgery, radiation and chemotherapy, the prognosis for patients affected by these diseases is often dismal.
The clinical signs that lead to diagnosis of gliomas include seizures, an increasing intracranial pressure leading to leakage of the blood-brain-barrier and oedema, which provokes nausea, vomiting, and headache, and progressive neurological and cognitive deficits. Magnetic Resonance Imaging (MRI) is then useful to identify the location and features of the tumour mass. The classification and grading of the type of glioma is possible after histological examination of a tumour sample, and is based on the hypothetical line of differentiation of the tumour cell. According to the World Health Organization (WHO) classification, gliomas include tumours composed mainly by ependymal cells (ependymomas), oligodendrocytes (oligodendrogliomas), mixtures of different glial cells (mixed gliomas, for instance oligoastrocytomas) and astrocytes (astrocytomas).
Astrocytoma
Classification and Histopathology
Among gliomas, the most common are astrocytomas, which are composed of astrocyte-like cells and often arise in the cerebrum. In children, they occur mainly in the cerebellum, in brain stem, and optic chiasma. Astrocytomas include tumours with highly variable natural behaviour and prognosis. They are usually classified in 4 grades (see Table 1) mainly on the basis of histopathological parameters.
WHO grade I astrocytoma (pilocytic astrocytoma) is more frequent in children, generally has low proliferative potential and is cured by surgery in most cases.
WHO grade II astrocytomas (diffuse astrocytoma) are poorly defined tumours that infiltrate the normal brain tissue, with isolated tumoral cells often observed at extraordinary distance from the primary tumour. They show a moderate cellularity and sporadic nuclear atypia. Most patients with grade II astrocytoma will recur with a higher grade tumour within 6 to 8 years.
WHO grade III astrocytomas (anaplastic astrocytoma) are distinguished from grade II tumours by their greater cellularity, increased mitotic activity and nuclear pleomorphism (anaplasia). Despite some long-term survivors, the median survival of patients with grade III astrocytoma is around 3 years.
WHO grade IV astrocytomas (or glioblastomas) preferentially arise in the cerebral hemispheres of adults and are highly devastating tumours. Compared to anaplastic astrocytomas they are characterized by the additional presence of endothelial cell proliferation and necrosis. They may develop after a history of lower grade astrocytoma (secondary glioblastoma) or de novo (primary glioblastoma). The prognosis is dismal for both types, with a median survival around 1 year, despite some advances with combined treatments (irradiation and chemotherapy).
TABLE 1WHO classification and grade criteria for astrocytoma.WHOGradeWHO DesignationHistological CriteriaIPilocytic AstrocytomaIIDiffuse AstrocytomaOne criterion: usually nuclear atypiaIIIAnaplasticTwo criteria: usually nuclear atypia andAstrocytomamitotic activityIVGlioblastomaThree criteria: nuclear atypia, mitosis,endothelial proliferation and/or necrosisPrimary and Secondary Glioblastoma
As mentioned above, two subtypes of glioblastoma are distinguished on the basis of their clinical features: primary glioblastoma, which develop very rapidly, usually in elderly people, without any evidence of pre-existing lesions, and secondary glioblastoma, which derives from the malignant evolution of previously diagnosed low grade astrocytoma and is more frequent in young patients. Histologically these two tumour types are indistinguishable and the clinical outcome is also similar, but genetically they are different.
Secondary Glioblastoma
Secondary glioblastomas in their initial phase as low grade astrocytomas are characterised by the frequent disruption of the TP53 locus (over 60% of grade II astrocytomas), with mutations in the gene (65%) and Loss of Heterozygosity (LOH) on the chromosome (17p13). Disruption of the p53 pathway dismantles two important cellular processes, as p53 functions as tumour suppressor by controlling cell cycle progression at the G1/S and G2/M checkpoints and apoptosis in response to several extracellular stimuli including DNA damage. Therefore a non-functional p53 pathway confers a growth advantage and genomic instability to astrocytoma cells, although alone is not sufficient for astrocytoma initiation.
The additional genetic alterations found in low grade astrocytomas that seem to be necessary for their initiation involve activation of Receptor Tyrosine Kinases (RTKs) in order to render astrocytoma cells independent from growth factors for their survival. A crucial mediator of RTKs signalling is the c-Src non-receptor tyrosine kinase. c-Src directly associate to RTKs activated by the binding of their corresponding growth factors and is concomitantly activated thereby synergising and cooperating in the stimulation of the multiple pathways activated by RTKs.
Primary Glioblastoma
Primary glioblastomas show deregulation of the same genetic pathways as secondary glioblastomas, namely the p53, the RTK-Ras, the RB and the AKT pathways, but they use different mechanisms.
In primary glioblastomas the p53 pathway is not disrupted by direct mutations of the TP53 gene, but rather through amplification and overexpression of the MDM2 gene (10% and 50%, respectively) and loss of p19 (40%). MDM2 functions as a negative regulator of the p53 transcriptional activity by targeting it for ubiquitin-mediated degradation through direct protein-protein binding. Furthermore MDM2 transcription is induced by p53 thus establishing a negative feedback loop that regulates the activity of both proteins. P19 prevents p53 degradation by directly binding to MDM2. p19 is encoded in the same CDKN2A gene as p16 by an alternative reading frame (ARF, another name of p19). This might explain why LOH of the CDKN2A locus on chromosome 9p21 is very frequent in primary glioblastomas (˜40%) since it disrupts two pathways at the same time: the p53 and the RB pathways. Whether the simultaneous deregulation of two crucial pathways for gliomagenesis might be one of the reasons for the extremely rapid development of primary glioblastoma remains to be clarified.
Angiogenesis and Anti-angiogenic Therapy of Glioma
Gliomas are among the most vascularised of human cancers. The process of angiogenesis, that is the formation of new vessels by sprouting of pre-existing ones or incorporation of endothelial cell progenitors, is critical for a tumour to develop. Without supply of oxygen and nutrients cancer cells cannot sustain all their malignant activities including indiscriminate growth, survival and invasion. Therefore, induction of an active production of new blood vessels is a crucial step during tumour progression.
In gliomas, control of angiogenesis is disturbed. There is often a poorly functional vasculature incapable of adequate oxygen supply inside the tumour resulting in the generation of large regions of low oxygen tension and subsequent necrosis. The presence of hypoxic areas in the tumour is at the origin of a vicious cycle, where hypoxia stimulates the formation of new blood vessels and these, given their structural and functional abnormalities are unable to provide sufficient oxygen supply, thereby causing new hypoxia to form.
Anti-angiogenic therapies have been proposed for the treatment of glioma (Tuettenberg et al., 2006). It has been noted, however, that inhibition of angiogenesis, while inhibiting glioma growth, leads to a substantial increase in glioma invasiveness (Lamszus et al., 2003), thus limiting the applicability of such approach. It has therefore been proposed to combine anti-angiogenic therapy of glioma with a therapy that inhibits gloma spreading (Lamszus et al., 2005).
Spreading
One of the main reasons accounting for glioma and astrocytoma lethality is the property of such tumours to spread. Astrocytomas very rarely metastasize outside the CNS, but the invasive cells remaining after surgical resection, regardless of its extension, will inevitably spread everywhere within the brain and lead to recurrence with a significant contribution to the demise of the patients. Already several decades ago neurosurgeons realised that even a radical resection, as hemispherectomy could not prevent astrocytoma recurrence and improve the survival of the patient. Astrocytoma cells can be found at large distances from the core lesion (several centimeters) and mainly disseminate as single cells in an integrin-dependent mechanism of migration defined as mesenchymal. They typically invade throughout the brain following three main anatomical structures: (i) the myelinated fiber tracts, usually of the corpus callosum; (ii) the abluminal surface of blood vessels and (iii) the glia limitans, the structure formed by astrocytes foot processes underneath the pia mater at the periphery of the brain parenchyma. The reason for these preferences are not yet clear, but the composition of the substrates supporting glioma cell migration is very likely to be of relevance.
Invasion of glioma cells requires the activation of several cellular processes. First, the cells need to adhere to the extracellular matrix. This step is important because the simultaneous link of membrane proteins with the extracellular environment and the cell cytoskeleton provides the cells the necessary force for traction and motility. Second, the cells have to detach from adjacent cells by disrupting their junctions. Third, to generate the space for movement, the cells have to locally degrade the extracellular matrix (ECM). This is accomplished through the release by glioma cells of active proteases. Fourth, an entire rearrangement of the cytoskeleton is needed for the cell to extend protrusions, lamellipodia and filopodia, establish adhesions in the direction of migration and finally retract its cell body. All these cell behaviors are orchestrated by an array of molecules; interaction with the ECM is mainly mediated by integrins establishing focal contacts, cell detachment from neighboring cells requires disruption of cadherins cell-cell junctions, degradation of the ECM is accomplished by metalloproteinases (MMPs) and the urokinase-plasminogen proteolytic system, and rearrangement of the cytoskeleton is governed by Rho family small GTPases.
In the US, approximately, 15,000 patients die from glioblastoma per year. The median survival time does not exceed 15 months. In view of the poor prognosis of glioma and in particular anaplastic astrocytoma and glioblastoma and the inavailibity of efficient treatment there is thus a need for new drug targets and compounds for the treatment of said disorders. In particular there is a need for compounds that inhibit growth of glioma and in particular anaplastic astrocytoma and glioblastoma. There is a need for compounds that inhibit or reduce spreading of glioma and in particular anaplastic astrocytoma and glioblastoma. New compounds for the treatment of glioma must be able to cross the blood-brain barrier. Additionally new targets and compounds for the treatment of glioma must be very specific, as cross-reactivity with normal brain tissue may lead to neurotoxicity with intolerable side effects (Gerber and Laterra, 2007).
While monoclonal antibodies have become standard therapy for breast and colon cancer as well as other malignancies, they were only used experimentally in the treatment of glioma, which poses unique challenges to antibody therapies (Gerber and Laterra, 2007). In particular, the blood-brain barrier is considered to be a major problem for successful application of high molecular weight compounds such as antibodies or other proteins to the treatment of glioma and other brain tumours as such high molecular weight compounds generally do not cross this barrier (Pardridge, 2006). The anti-cancer antibody trastuzumab, for example, fails to cross the blood-brain barrier leading to brain metastasis of breast cancer patients (Lai et al., 2004; Bendell et al., 2003). As anti-tumour antibodies that were systemically delivered had no therapeutic effect, while being effective when locally administered into the brain, local delivery of large molecular entities such as antibodies for the treatment of glioma has been the mainstay of therapy (Sampson et al., 2000).
Junctional Adhesion Molecules (JAMs)
General Features
Junctional Adhesion Molecules (JAMs) are a family of proteins belonging to the immunoglobulin Superfamily (IgSf) class of adhesion molecules. They are generally localised at sites of cell-cell contacts and particularly abundant in tight junctions, the specialised cellular structures that keep cell polarity and serve as barriers to prevent the diffusion of molecules across intercellular spaces and along the basolateral-apical regions of the plasma membrane.
Several human and mouse Junctional Adhesion Molecules (JAMs) have been simultaneously cloned in different laboratories and the amino and nucleic acid sequences published. This has raised confusion in their nomenclature which was the subject of a complete revision (Muller, 2003; Mandell and Parkos, 2005). The current nomenclature described in Table 2 will be used herein.
TABLE 2JAM nomenclatureCurrentPreviousNomenclatureNomenclatureSpeciesJAM-AJAM/JAM-1MouseJAM/JAM-1HumanF11 ReceptorHumanJAM-BJAM-3MouseVE-JAMMouse, HumanJAM2HumanJAM-CJAM-2MouseJAM3HumanJAM-3HumanAmong the family members JAM-A, JAM-B, and JAM-C share a similar protein structure with 31-36% amino acid identity, and molecular masses ranging from 40-45 kDa. JAMs are characterised by two extracellular immunoglobulin domains, one membrane distal of VH-type and one membrane proximal of C2-type, a trans-membrane domain and a cytoplasmic domain. The extracellular domains contain several putative N-glycosylation sites.
In addition, the VH-type Ig domain mediates cis dimerisation of JAM-A and trans homotypic associations between JAM-A dimers emanating from opposing cells. The cis dimerisation motif is conserved in JAM-B and JAM-C which dimerise and trans-interact in a similar way (Lamagna et al., 2005b).
The cytoplasmic domains contain potential tyrosine as well as serine/threonine phosphorylation sites and are characterised by the presence of type II PDZ-binding motifs. PDZ binding motifs are often found in scaffolding proteins where they mediate protein-protein interactions, and the first proteins identified with such structures were PSD-95, Discs-large A and Zonula Occludens-1, hence the name PDZ. Additional JAM proteins representing a distinct subfamily containing type I PDZ binding motifs have recently been described. These JAMs include Coxsackie and adenovirus Receptor (CAR), Endothelial cell-selective Adhesion Molecule (ESAM), and JAM-4. However, the presence of a diverse type of PDZ binding motif suggests that they might interact with diverse sets of intracellular partner molecules and exert different functions.
JAM-A, JAM-B and JAM-C have different patterns of tissue and cell distribution, intracellular and extracellular molecular partners and functions.
TABLE 3Expression of JAM-A, JAM-B and JAM-C in mouse and human.MouseHumanJAM-AEndothelial cellsEndothelial cellsEpithelial cellsEpithelial cellsPlateletsPlateletsDendritic cellsMonocytesLymphocytesNeutrophilsJAM-BEndothelial cellsEndothelial cellsLymphaticLymphaticEndothelial cellsEndothelial cellsJAM-CEndothelial cellsEndothelial cellsLymphaticLymphaticEndothelial cellsEndothelial cellsSpermatidsEpithelial cellsPlateletsDendritic cellsLymphocytes
TABLE 4Extracellular ligands of JAM-A, JAM-B and JAM-C.JAM ligandsIntegrin ligandsJAM-AJAM-ALFA-1 (CD11a/CD18, αLβ2)reovirusαVβ3JAM-BJAM-BVLA-4 (α4β1)JAM-CJAM-CJAM-BMAC-1 (αMβ2, CD11b/CD18)JAM-Cp150, 95 (αXβ2, CD11c/CD18)CARJAM-B
JAM-B expression has been shown in endothelial cells and lymphatic endothelial cells in both mouse and human. JAM-B expression was detected on human endothelial cells in inflammatory sites and tumour foci. In the brain, Northern blot analysis revealed a weak expression of JAM-B mRNA, but no analysis was performed at the protein level, and the cell types expressing JAM-B in brain were not identified (Palmeri et al., 2000).
The cellular localisation of JAM-B seems to differ from that of other family members. JAM-B does not appear to be situated in tight junction structures but rather to be more diffusely distributed on the plasma membrane, as demonstrated by the ectopic expression of JAM-B in Madin-Darby canine kidney (MDCK) epithelial cells.
JAM-B can establish both homophilic trans-interactions and heterophilic associations with JAM-C. It appears that these heterophilic bindings mediate interaction of JAM-B on endothelial cells with JAM-C on leukocytes and platelets. JAM-B also interacts with integrin α4β1 and this binding seems to require prior engagement of JAM-C.
JAM-C
JAM-C expression has been observed in both mouse and human endothelial cells and lymphatic endothelial cells, and in several human leukocytes and platelets. In addition, JAM-C was found in human intestinal epithelia and in mouse spermatids. In the human brain, by Northern blot analysis JAM-C mRNA was expressed in several regions, but no investigations of protein expression and of the cell types expressing JAM-C was performed (Arrate et al., 2001).
JAM-C is localised in tight junctions by confocal analysis and co-distribution with the known tight junction protein occludin (Aurrand-Lions et al., 2001a). However, in intestinal human epithelia JAM-C was detected in the basolateral membrane of the cells in desmosomal structures (Zen et al., 2004), suggesting the existence of cell-type specific sub-cellular localisation of JAM-C and consequently potential different functions for this protein.
JAM-C can engage in homotypic and, with higher affinity, in heterotypic trans-interactions with JAM-B (Lamagna et al., 2005b). In addition, other ligands for JAM-C include integrins αMβ2 (MAC-1, CD11b/CD18) and αXβ2 (p150/95, CD11c/CD18) (Chavakis et al., 2004; Santoso et al., 2002; Zen et al., 2004) and CAR (Mirza et al., 2006).
To date JAM-C has been implicated in leukocyte trafficking, angiogenesis and cell polarity. It has been shown that anti-JAM-C antibodies block lymphocyte trans-endothelial migration in vitro (Johnson-Leger et al., 2002a), and that JAM-C promotes neutrophil trans-endothelial migration in vitro and in vivo in a αMβ2 dependent manner.
The role of JAM-C in angiogenesis has been demonstrated both in vitro and in vivo (Imhof and Aurrand-Lions, 2000; Imhof and Aurrand-Lions, 2005; Lamagna et al., 2005a). In particular, an antibody against JAM-C was shown to block the in vitro outgrowth of blood vessels in an aortic ring assay. In addition, an anti-JAM-C was shown to block the in vitro outgrowth of blood vessels in an aortic ring assay. In addition, an anti-JAM-C antibody inhibited in vivo growth and reduced tumour associated angiogenesis of a Lewis Lung Carcinoma (LLC) mouse tumour model (Lamagna et al., 2005a). The reduction in tumour-associated angiogenesis was attributed to a decreased recruitment of macrophages to the tumour bed, since the anti-JAM-C antibody had no effect on endothelial cell proliferation and apoptosis in vitro.
WO 2006/084078 reports on immunohistochemistry staining of different tumour tissues with the murine anti-human JAM-C antibodies PACA4 and LUCA14. The staining pattern was heterogenous with no consistent positive staining of the tumour tissues tested with PACA4 and LUCA14. Various normal and tumour cell lines were also tested for JAM-C expression with PACA4 and LUCA14. A number of glioma-derived cell lines stained weakly positive or positive with only PACA4. The staining pattern on non-tumour brain tissue; i.e. astrocytes, however, was not reported. It thus, remains unclear whether JAM-C is expressed highly and specifically on astrocytoma or glioblastoma, in order to be a suitable target for the treatment of glioma. Additionally it remains unclear whether anti-JAM-C antibodies or other high molecular weight compounds specifically binding to JAM-C would cross the blood-brain barrier in order to be effective in the treatment of glioma or other brain tumours when administered systemically.